Atypical soluble guanylyl cyclases control brain size in Drosophila

Hypoxia-induced proliferation of neural stem cells has a crucial role in brain development. In the brain of Drosophila melanogaster , the optic lobe exhibits progressive hypoxia during larval development. Here, we investigate an alternative oxygen-sensing mechanism within this brain compartment, distinct from the canonical hypoxia signaling pathway mediated by HIF. Using genetic tools, immunostaining, and confocal microscopy, we demonstrate that the loss of the atypical soluble guanylyl cyclase (asGC) subunit Gyc88E , or the ectopic expression of Gyc89Db in neural stem cells leads to increased optic lobe volume. We propose the existence of a link between cGMP signaling and neurogenesis in the developing brain.


Description
Oxygen availability is a powerful driver of evolutionary novelty, and metazoans have developed sophisticated hypoxia sensing systems that are intricately related to the control of stem cell niches and development (Simon and Keith 2008;Rytkönen et al. 2011;Hammarlund et al. 2018).Increased atmospheric oxygen levels during evolution had profound effects on insects enabling them, for instance, to develop larger body size and the ability to fly (Stamati et al. 2011).
The atypical soluble guanylyl cyclase (asGC) subunits Gyc88E, Gyc89Da and Gyc89Db are expressed from embryo to adult stages in Drosophila melanogaster (Langlais et al. 2004;Morton et al. 2005).They are regulated by O 2 levels and when activated by hypoxia generate cGMP, functioning as molecular O 2 sensors (Vermehren et al. 2006) with faster responses than the canonical hypoxia pathway (de Lima et al. 2021).
The neural stem cells within the optic lobe (OL) of the Drosophila larval brain are hypoxic relative to the central brain (Misra et al. 2017), with different cell types experiencing varying degrees of hypoxia (Baccino-Calace et al. 2020).There is evidence that optic lobe progenitor cells might not exhibit a canonical hypoxia response (Baccino-Calace et al. 2020).In situ hybridization data (Langlais et al. 2004) suggests that asGC subunits are expressed within the OL, potentially linking O 2 sensing to an alternative signaling pathway activated by hypoxia.Furthermore, transcriptomic data analysis (Yang et al. 2015), revealed that Gyc89Db is expressed in L3 neuroblasts.The combination of different asGC subunits might provide different sensitivity thresholds to hypoxia to Drosophila neurons (Lu et al. 2024).
Here, we used genetic tools to investigate the role of asGC in larval brain development.We explored asGC loss-of-function using a Gyc88E -/-mutant and a Gyc89Da -/-Db -/-double mutant.Additionally, we performed targeted gain-of-function with drivers specific to ectopically express Gyc89Db in neuroepithelial cells or overexpressing it in neuroblasts.
As the hypoxic OL remains neurogenic during larval life, we hypothesized that this hypoxia might activate asGC.Given that cGMP derived from nitric oxide signaling can activate mammalian neural stem cell proliferation (Santos et al. 2014), if hypoxia-driven cGMP signaling were to activate neural stem cell proliferation, loss of asGC function would abolish it.Contrary to our expectation, the Gyc88E -/-loss-of-function mutant exhibited a 1.9-fold increase in brain hemisphere volume (Figure 1A).Interestingly, the double mutation Gyc89Da -/-Db -/-had not the same effect.Notably, ectopic expression in NE or overexpression of Gyc89Db in NB lead to an increase in overall brain size (Figure 1A).The larval brain comprises two functionally and developmentally different compartments, the central brain (CB) which is formed mostly during embryonic stages and contains the neuronal synapses and the optic lobe (OL), which lack fully differentiated neurons with synapses and remains neurogenic throughout larval stages (Hartenstein et al. 2008;Baccino-Calace et al. 2020).
When we measured and compared CB and OL volumes separately, we observed that the macrocephalous phenotype observed in Gyc88E -/-mutants can be explained by a specific effect on the CB.Gyc88E -/-loss-of-function mutants showed a 2.1-fold increase in CB volume (Figure 1B), while the OL volume remains unaffected (Figure 1C).The double mutation Gyc89Da -/- Db -/-also elicited an increase in CB volume but limited to a lesser extent (1.7-fold, Figure 1B).
Ectopic expression of Gyc89Db under the control of the OL-specific neuroepithelial driver GAL4 c855a (Egger et al. 2007) resulted in increased volume of both CB (Figure 1B) and OL (Figure 1C).Overexpression under the pan-neuroblast driver inscuteable-GAL4 reproduced the macrocephalous phenotype only in the CB (Fig. 1B).Provided the OL is the major neurogenic region within the larval brain, experiencing a dramatic volume increase between 24-72 hs after larval hatching (Baccino-Calace et al. 2020), we reasoned that, in the larva, an eventual increase in proliferation would only be observable in this hypoxic brain region.
Aiming to further investigate the cellular basis of the macrocephalous phenotype, we assessed neuroblast number and proliferation within the OL.asGC loss-of-function mutants and ectopic expression of Gyc89Db in the OL neuroepithelium increased neuroblast number in this region, while overexpression in neuroblasts did not (Figure 1D).
Despite the increased OL volume, no differences in the number of proliferating neuroblasts were observed in the loss-offunction mutants (Figure 1E), whereas Gyc89Db ectopic expression in neuroepithelial cells or overexpression in neuroblasts increased the number of mitotically active neuroblasts by 2.5-fold and 3.4-fold, respectively (Figure 1E).Furthermore, larvae with Gyc88E -/-loss of function or ectopic expression of Gyc89Db in the neuroepithelium or overexpression in neuroblasts show increased total cell number in mitosis in the optic lobe (Figure 1F), suggesting a link between oxygen-dependent cGMP signaling and the control of proliferation in neural progenitor cells.
Previous research has shown that Gyc88E is active in the absence of additional subunits, while Gyc89Da and Gyc89Db enhanced the activity of Gyc88E when co-expressed, suggesting that these enzymes acted likely as heterodimers (Morton and Vermehren 2007;Morton 2011).It is important to mention that Gyc89Db is expressed in the brain during larval development (Langlais et al. 2004) and produces cyclic GMP in response to low oxygen in vitro (Morton 2004;Vermehren et al. 2006).Thus, asGCs may fine tune the cellular response promoting proliferation through hypoxia-driven cGMP signaling.

Reagents
Stocks: Stocks obtained from the Bloomington Drosophila Stock Center (BDSC) or kindly provided by colleagues as stated under the Methods section.